Remedies for dry eye and diseases associated with dry eye

ABSTRACT

Remedies for dry eye and diseases associated with dry eye which contain as the active ingredient peptides represented by the general formula (I):
 
H-His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-X 1 -Gln-X 2 -Ala-Val-X 3 -X 4 -Tyr-Leu-X 5 -X 6 (SEQ ID NOS: 26-36)   (I)
 
wherein X 1 , X 3  and X 4  represent Lys or Arg, respectively; X 2  represents Met, Leu or nLeu; X 5  represents a chemical bond, Asn, Asn-Ser, Asn-Ser-Ile, Asn-Ser-Ile-Leu(SEQ ID NO: 37) or Asn-Ser-Ile-Leu-Asn-X 7 (SEQ ID NOS: 38-43) (wherein X 7  represents a chemical bond, Gly, etc.); and X 6  represents —OH or —NH 2 , or pharmaceutically acceptable salts thereof:.

This application is a U.S. national stage of International ApplicationNo. PCT/JP02/11490 filed Nov. 5, 2002.

TECHNICAL FIELD

The present invention relates to remedies for dry eye and diseasesassociated with dry eye.

BACKGROUND ART

Tears play an important role in maintaining the normal visual function.Tears cover the surface of cornea and conjunctiva to retain thewettability thereof and, at the same time, tears fill a depression dueto a microvillus on the corneal surface to make the surface smooth,therefore, it becomes possible to obtain a clear image. In addition,epithelial cells of cornea and conjunctiva actively metabolize cellularcomponents and unnecessary cells and metabolites are detached anddischarged from the most superficial surface, tears not only wash outthem but also supplement necessary oxygen and nutrients. Further, tearswash out foreign matters which intrude on the surface of cornea andconjunctiva, and play a role of defending infection against viruses,bacteria and fungi which have invaded from the outside by thebacteriostatic action of tears. Furthermore, tears work as a synoviabetween an eyelid and cornea and conjunctiva so that nictitating andeyeball movement are smoothly done. Thus, tears are a minor amount offluid for forming a slight thin film on the surface of cornea andconjunctiva, and are indispensable for maintaining the transparency andthe homeostatis of cornea by various elaborate mechanisms.

The state where a secretion disorder of tears causes abnormality on thesurface of cornea and conjunctiva is generally called dry eye. When adisorder of cornea and conjunctiva due to dry eye is caused, supplementof artificial tears, dropping of a viscoelastic substance having highmoisture retention such as hyaluronic acid into eyes, and use of dry eyespectacles for keeping the eye surface wet and ameliorating dry symptomare performed. However, while symptom can be ameliorated by thesesymptomatic treatment methods, these treatment methods are not anetiotropic method for fundamental treatment. Since it is thought thattears have the effect of curing corneal and conjunctival disorder due todry eye by their natural function as described above, a compound whichdirectly acts on lacrimal gland and promotes tear secretion is expectedto be a useful remedy for dry eye and diseases associated with dry eye.

Lacrimal glands are controlled by parasympathetic nerve and sympatheticnerve and the former is dominant. Parasympathetic nerve secretesacetylcholine and VIP (Vasoactive Intestinal Peptide). On the otherhand, sympathetic nerve secretes norepinephrine and neuropeptide Y.Acetylcholine, norepinephrine and VIP mainly stimulate lacrimal glands(Dartt D A et al., Adv Eep Med Biol 438: 113-121, 1998). Acetylcholineis shown to activate muscarinic cholinergic route and be involved alsoin lacrimal secretion (Nakamura M et al., Curr Eye Res 16: 614-619,1997). Norepinephrine is a sympathomimetic amine which binds toadrenaline α and β receptors, and secretes tear protein via an α₁adrenaline receptor (Dartt D A, Curr Eye Res 8:619-636, 1989). VIP is apeptide having various biological activities which relaxes a smoothmuscle of a digestive tract and a blood vessel, and it is reported thatreceptors for this VIP are present in lacrimal glands (Hodges R R etal., Invest Opthalmol Vis Sci 38:610-619, 1997), and actually promotessecretion of protein from lacrimal glands (Dartt D A et al., Am JPhysiol 247:G502-509, 1984).

As described above, distribution of receptors such as muscarine,norepinephrine or VIP in lacrimal glands, and further, participation inlacrimal secretion have been shown. However, currently, thesephysiologically active ingredients have not been put into practice yetas an agent for preventing or treating dry eye based on the lacrimalsecretion promoting activity. With respect to VIP, U.S. Pat. No.4,745,100 discloses a method of promoting lacrimal secretion by topicaladministration, but there is no specific description of VIP derivativestherein. In addition, peptides exhibiting excellent bronchodilatoractivities and the digestive tract movement inhibiting activities as VIPderivatives are disclosed in JP-A 8-333276 and JP-A 2001-151799,respectively, but there are no description regarding lacrimal secretionand dry eye in these publications.

OBJECTS OF THE INVENTION

An object of the present invention is to provide a remedy for dry eyeand diseases associated with dry eye by promotion of lacrimal secretion.

SUMMARY OF THE INVENTION

The present inventors studied intensively paying their attention to thelacrimal secretion promoting activity harbored by VIP and, as a result,found that VIP derivatives shown in JP-A 8-333276 and JP-A 2001-151799have an excellent lacrimal secretion promoting activity. They havefurther studied, which has resulted in completion of the presentinvention.

That is, the present invention provides:

(1) A remedy for dry eye or diseases associated with dry eye whichcomprises a peptide represented by the general formula (I):H-His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-X₁-Gln-X₂-Ala-Val-X₃-X₄-Tyr-Leu-X₅-X₆(SEQID NOS: 26-36)  (I)wherein X₁, X₃ and X₄ represent Lys or Arg, respectively; X₂ representsMet, Leu or nLeu; X₅ represents a chemical bond, Asn, Asn-Ser,Asn-Ser-Ile, Asn-Ser-Ile-Leu(SEQ ID NO: 37) orAsn-Ser-Ile-Leu-Asn-X₇(SEQ ID NOS: 38-43) (wherein X₇ represents achemical bond, Gly, Gly-Lys, Gly-Lys-Arg, Gly-Arg or Gly-Arg-Arg); X₆represents —OH or —NH₂, provided that, when X₁, X₃ and X₄ are Lys, X₅ isAsn-Ser-Ile-Leu-Asn-X₇(SEQ ID NOS: 38-43), X₇ is a chemical bond, and X₆is —NH₂, then X₂ represents Leu or nLeu, or a pharmaceuticallyacceptable salt thereof;

(2) The remedy according to the above (1), wherein X₅ isAsn-Ser-Ile-Leu-Asn-X₇(SEQ ID NOS: 38-43), and X₇ is Gly, Gly-Lys,Gly-Lys-Arg, Gly-Arg or Gly-Arg-Arg in the general formula (I).

(3) The remedy according to the above (1), wherein X₅ is a chemicalbond, Asn, Asn-Ser, Asn-Ser-Ile, Asn-Ser-Ile-Leu(SEQ ID NO: 37) orAsn-Ser-Ile-Leu-Asn-X₇(SEQ ID NOS: 38-43) (wherein X₇ is a chemicalbond) in the general formula (I);

(4) The remedy according to the above (1), wherein X₁, X₃ and X₄ areArg, X₂ is Leu, X₅ is Asn-Ser-Ile-Leu-Asn-X₇ (SEQ ID NOS: 38-43), X₇ isGly-Arg-Arg, and X₆ is —NH₂ in the general formula (I);

(5) The remedy according to the above (1), wherein X₁, X₃ and X₄ areLys, X₂ is Leu, X₅ is a chemical bond, and X₆ is —NH₂ in the generalformula (I);

(6) The remedy according to any one of the above (1) to (5), which is apreparation topically administered to the eye;

(7) The remedy according to the above (6), wherein the preparationtopically administered to the eye is eye drops;

(8) A pharmaceutical composition for treating dry eye or diseasesassociated with dry eye, which comprises a peptide represented by thegeneral formula (I):H-His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-X₁-Gln-X₂-Ala-Val-X₃-X₄-Tyr-Leu-X₅-X₆(SEQID NOS: 26-36)  (I)wherein X₁, X₃ and X₄ represent Lys or Arg, respectively; X₂ representsMet, Leu or nLeu; X₅ represents a chemical bond, Asn, Asn-Ser,Asn-Ser-Ile, Asn-Ser-Ile-Leu (SEQ ID NO: 37) orAsn-Ser-Ile-Leu-Asn-X₇(SEQ ID NOS: 38-43) (wherein X₇ represents achemical bond, Gly, Gly-Lys, Gly-Lys-Arg, Gly-Arg or Gly-Arg-Arg); X₆represents —OH or —NH₂, provided that, when X₁, X₃ and X₄ are Lys, X₅ isAsn-Ser-Ile-Leu-Asn-X₇(SEQ ID NOS: 38-43), X₇ is a chemical bond, and X₆is —NH₂, then X₂ represents Leu or nLeu, or a pharmaceuticallyacceptable salt thereof and a pharmaceutically acceptable carrier;

(9) The pharmaceutical composition according to the above (8), whereinX₅ is Asn-Ser-Ile-Leu-Asn-X₇(SEQ ID NOS: 38-43), and X₇ is Gly, Gly-Lys,Gly-Lys-Arg, Gly-Arg or Gly-Arg-Arg in the general formula (I);

(10) The pharmaceutical composition according to the above (8), whereinX₅ is a chemical bond, Asn, Asn-Ser, Asn-Ser-Ile, Asn-Ser-Ile-Leu(SEQ IDNO: 37) or Asn-Ser-Ile-Leu-Asn-X₇(SEQ ID NOS: 38-43) (wherein X₇ is achemical bond) in the general formula (I);

(11) The pharmaceutical composition according to the above (8), whereinX₁, X₃ and X₄ are Arg, X₂ is Leu, X₅ is Asn-Ser-Ile-Leu-Asn-X₇(SEQ IDNOS: 38-43), X₇ is Gly-Arg-Arg, and X₆ is —NH₂ in the general formula(I);

(12) The pharmaceutical composition according to the above (8), whereinX₁, X₃ and X₄ are Lys, X₂ is Leu, X₅ is a chemical bond, and X₆ is —NH₂in the general formula (I);

(13) The pharmaceutical composition according to any one of the above(8) to (12), which is a composition topically administered to the eye;

(14) The pharmaceutical composition according to the above (13), whereinthe composition topically administered to the eye is eye drops;

(15) Use of a peptide represented by the general formula (I):H-His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-X₁-Gln-X₂-Ala-Val-X₃-X₄-Tyr-Leu-X₅-X₆(SEQID NOS: 26-36)  (I)wherein X₁, X₃ and X₄ each represent Lys or Arg, respectively; X₂represents Met, Leu or nLeu; X₅ represents a chemical bond, Asn,Asn-Ser, Asn-Ser-Ile, Asn-Ser-Ile-Leu (SEQ ID NO: 37) orAsn-Ser-Ile-Leu-Asn-X₇(SEQ ID NOS: 38-43) (wherein X₇ represents achemical bond, Gly, Gly-Lys, Gly-Lys-Arg, Gly-Arg or Gly-Arg-Arg); X₆represents —OH or —NH₂, provided that, when X₁, X₃ and X₄ are Lys, X₅ isAsn-Ser-Ile-Leu-Asn-X₇(SEQ ID NOS: 38-43), X₇ is a chemical bond, and X₆is —NH₂, then X₂ represents Leu or nLeu, or a pharmaceuticallyacceptable salt thereof for preparing a medicament for treating dry eyeor diseases associated with dry eye;

(16) The use according to the above (15), wherein X₅ isAsn-Ser-Ile-Leu-Asn-X₇(SEQ ID NOS: 38-43), and X₇ is Gly, Gly-Lys,Gly-Lys-Arg, Gly-Arg or Gly-Arg-Arg in the general formula (I);

(17) The use according to the above (15), wherein X₅ is a chemical bond,Asn, Asn-Ser, Asn-Ser-Ile, Asn-Ser-Ile-Leu(SEQ ID NO: 37) orAsn-Ser-Ile-Leu-Asn-X₇(SEQ ID NOS: 38-43) (wherein X₇ is a chemicalbond) in the general formula (I);

(18) The use according to the above (15), wherein X₁, X₃ and X₄ are Arg,X₂ is Leu, X₅ is Asn-Ser-Ile-Leu-Asn-X₇ (SEQ ID NOS: 38-43), X₇ isGly-Arg-Arg, and X₆ is —NH₂ in the general formula (I);

(19) The use according to the above (15), wherein X₁, X₃ and X₄ are Lys,X₂ is Leu, X₅ is a chemical bond, and X₆ is —NH₂ in the general formula(I);

(20) The use according to any one of the above (15) to (19), wherein themedicament is a medicament topically administered to the eye;

(21) The use according to the above (20), wherein the medicamenttopically administered to the eye is eye drops;

(22) A method for treating dry eye or diseases associated with dry eye,which comprises administering an effective amount of a peptiderepresented by the general formula (I):H-His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-X₁-Gln-X₂-Ala-Val-X₃-X₄-Tyr-Leu-X₅-X₆(SEQID NOS: 26-36)  (I)wherein X₁, X₃ and X₄ represent Lys or Arg, respectively; X₂ representsMet, Leu or nLeu; X₅ represents a chemical bond, Asn, Asn-Ser,Asn-Ser-Ile, Asn-Ser-Ile-Leu(SEQ ID NO: 37) orAsn-Ser-Ile-Leu-Asn-X₇(SEQ ID NOS: 38-43) (wherein X₇ represents achemical bond, Gly, Gly-Lys, Gly-Lys-Arg, Gly-Arg or Gly-Arg-Arg); X₆represents —OH or —NH₂, provided that, when X₁, X₃ and X₄ are Lys, X₅ isAsn-Ser-Ile-Leu-Asn-X₇(SEQ ID NOS: 38-43), X₇ is a chemical bond, and X₆is —NH₂, then X₂ represents Leu or nLeu, or a pharmaceuticallyacceptable salt thereof to a warm-blooded animal in need of treatment ofdry eye or diseases associated with dry eye;

(23) The method according to the above (22), wherein X₅ isAsn-Ser-Ile-Leu-Asn-X₇(SEQ ID NOS: 38-43), and X₇ is Gly, Gly-Lys,Gly-Lys-Arg, Gly-Arg or Gly-Arg-Arg in the general formula (I);

(24) The method according to the above (22), wherein X₅ is a chemicalbond, Asn, Asn-Ser, Asn-Ser-Ile, Asn-Ser-Ile-Leu(SEQ ID NO: 37) orAsn-Ser-Ile-Leu-Asn-X₇(SEQ ID NOS: 38-43) (wherein X₇ is a chemicalbond) in the general formula (I);

(25) The method according to the above (22), wherein X₁, X₃ and X₄ areArg, X₂ is Leu, X₅ is Asn-Ser-Ile-Leu-Asn-X₇(SEQ ID NOS: 38-43), X₇ isGly-Arg-Arg, and X₆ is —NH₂ in the general formula (I); and

(26) The method according to the above (22), wherein X₁, X₃ and X₄ areLys, X₂ is Leu, X₅ is a chemical bond, and X₆ is —NH₂ in the generalformula (I).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows secretion rates of protein from isolated lacrimals gland ofrabbits when VIP, Peptide 5 or Peptide 15 is reacted with the lacrimalgland.

FIG. 2 shows an increment of lacrimal secretion amounts (Schirmer'svalue) with time, when VIP or Peptide 5 is applied to the eyes of arabbit once.

DETAILED DESCRIPTION OF THE INVENTION

The VIP derivative used in treatment of dry eye and diseases associatedwith dry eye of the present invention is a peptide represented by thegeneral formula (I), and X₁, X₃ and X₄ are Lys or Arg. X₂ is Met, Leu ornLeu, preferably Leu. X₅ can be a chemical bond, Asn, Asn-Ser,Asn-Ser-Ile, Asn-Ser-Ile-Leu(SEQ ID NO: 37) orAsn-Ser-Ile-Leu-Asn-X₇(SEQ ID NOS: 38-43), preferably a chemical bond orAsn-Ser-Ile-Leu-Asn-X₇(SEQ ID NOS: 38-43). When X₅ isAsn-Ser-Ile-Leu-Asn-X₇(SEQ ID NOS: 38-43), X₇ is a chemical bond, Gly,Gly-Lys, Gly-Lys-Arg, Gly-Arg or Gly-Arg-Arg, most preferablyGly-Arg-Arg. X₆ is —OH or —NH₂, more preferably —NH₂.

Representative examples of the VIP derivative represented by the generalformula (I) of the present invention include peptides 1 to 25 in Table 1(referred to as Peptides 1 to 25, respectively), and the like. These arepeptides corresponding to SEQ ID NOs: 1 to 25 in Sequence Listinghereinafter. Inter alia, Peptide 5 and Peptide 15 are advantageouslyused.

Peptides 1 to 25 (corresponding to SEQ ID NOs: 1 to 25 shown in SequenceListing)H-His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-X₁-Gln-X₂-Ala-Val-X₃-X₄-Tyr-Leu-X₅(—X₇)—X₆(SEQID NOS: 26-36)  (I)

TABLE 1 Peptide X₁ X₂ X₃ X₄ X₅ X₇ X₆* 1 Arg Leu Arg Arg Asn-Ser-Ile- Gly-NH₂ Leu-Asn 2 Arg Leu Arg Arg Asn-Ser-Ile- Gly-Lys -NH₂ Leu-Asn 3 ArgLeu Arg Arg Asn-Ser-Ile- Gly-Arg -NH₂ Leu-Asn 4 Arg Leu Arg ArgAsn-Ser-Ile- Gly-Lys- -NH₂ Leu-Asn Arg 5 Arg Leu Arg Arg Asn-Ser-Ile-Gly-Arg- -NH₂ Leu-Asn Arg 6 Arg Leu Arg Arg Asn-Ser-Ile- Gly-Lys- -OHLeu-Asn Arg 7 Lys Met Lys Lys Asn-Ser-Ile- -OH Leu-Asn 8 Lys Met Lys LysAsn-Ser-Ile- -NH₂ Leu 9 Lys Met Lys Lys Asn-Ser-Ile -NH₂ 10 Lys Met LysLys Asn-Ser -NH₂ 11 Lys Met Lys Lys Asn -NH₂ 12 Lys Met Lys Lys-(chemical -NH₂ bond) 13 Lys Leu Lys Lys Asn-Ser -NH₂ 14 Lys Leu Lys LysAsn -NH₂ 15 Lys Leu Lys Lys -(chemical -NH₂ bond) 16 Arg Met Arg ArgAsn-Ser-Ile- -NH₂ Leu 17 Arg Met Arg Arg Asn-Ser-Ile -NH₂ 18 Arg Met ArgArg Asn-Ser -NH₂ 19 Arg Met Arg Arg Asn -NH₂ 20 Arg Met Arg Arg-(chemical -NH₂ bond) 21 Arg Leu Arg Arg Asn-Ser-Ile- -NH₂ Leu 22 ArgLeu Arg Arg Asn-Ser-Ile -NH₂ 23 Arg Leu Arg Arg Asn-Ser -NH₂ 24 Arg LeuArg Arg Asn -NH₂ 25 Arg Leu Arg Arg -(chemical -NH₂ bond) *X₆ indicatesa carboxyl terminal of an amino acid sequence in Sequence Listinghereinafter.

Examples of a pharmaceutically acceptable salt of the compound of thegeneral formula (I) include salts with an alkali metal such as sodium,potassium and the like; salts with an alkaline earth metal such ascalcium, magnesium and the like; salts with an inorganic base such as analuminum salt, an ammonium salt and the like; salts with an organic basesuch as trimethylamine, pyridine, picoline, ethanolamine,diethanolamine, triethanolamine, dicyclohexylamine,N,N-dibenzylethylenediamine and the like; salts with an inorganic acidsuch as hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid,phosphoric acid and the like; salts with an organic acid such as formicacid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid,tartaric acid, lactic acid, maleic acid, citric acid, succinic acid,malic acid, methanesulfonic acid, benzenesulfonic acid,p-toluenesulfonic acid and the like; and salts with a polymer acid suchas tannic acid, carboxymethylcellulose, polylactic acid, polyglycolicacid and the like.

The VIP derivative of the general formula (I) can be synthesizedaccording to a known conventional method for synthesizing a peptide asshown, for example, in JP-A 8-333276, JP-A 9-100237, JP-A 11-100399,JP-A 2001-151799 and JP-A 2001-226284.

The VIP derivative of the general formula (I) has the followingproperties.

First, the VIP derivative has high stability of a molecule in a livingbody. Although peptides and proteins are rapidly metabolized bypeptidase in a living body, the peptides having increased basicity amongthe VIP derivatives used in the present invention exhibit resistance tometabolism. That is, since the basicity is increased by substituting oneor more of 15^(th), 20^(th) and 21^(st) lysines among amino acidsconstituting VIP with arginine, and adding a basic amino acid such aslysine or arginine following a glycine residue on a C-terminal side, thestrong affinity with acidic polysaccharides which are present in anextremely large amount in a living body is exhibited. By this nature,resistance to degradation by an endopeptidase such as trypsin isincreased in a living body. For example, in a trypsin digestion testusing Peptide 5, a higher remaining rate than that of VIP is shown inthe presence of chondroitin sulfate (Test Example 1). This tendency isdemonstrated more clearly in a peptide digestion test using abronchoalveolar lavage fluid, and the remaining rate of Peptide 5 after90 minutes is about 1.8-fold as higher as that of VIP (Test Example 2).Due to the aforementioned excellent stability in a living body, thesehighly basic VIP derivatives have a long-lasting pharmacologicalactivity. In addition, when topically applied to eyes, they can be usedas an eye drop preparation which hardly undergoes enzymatic degradationafter application to eyes. For example, it is demonstrated in TestExample 5 that application of Peptide 5 to eyes has a longer-lastinglacrimal secretion promoting activity than that of VIP.

In addition, although 17^(th) methionine in amino acid residuesconstituting VIP is easily oxidized, the VIP derivative in which thisplace is substituted with leucine or norleucine exhibits resistance tooxidation. Therefore, among the VIP derivatives used in the presentinvention, a 17-leucine-substituted peptide is hardly oxidized, and canbe used as stable eye drops.

In the present invention, the VIP derivative of the general formula (I)or a pharmaceutically acceptable salt thereof (hereinafter, referred toas “VIP derivative” in some cases) is systemically or topicallyadministered as a remedy for dry eye and diseases associated with dryeye (hereinafter, abbreviated as “dry eye remedy” in some cases)Systemically, they are administered parenterally (administered as aninjectable preparation such as intravenus injection, subcutaneousinjection and intramuscular injection, or as a suppository) and orally.Topically, they are administered to skin or eyes.

Examples of a dosage form of a preparation, which is administeredparenterally, include injectable preparations, suppositories, and thelike. When formulated into an injectable preparation, for example, asolvent (distilled water for injection), a stabilizer (sodium edetateetc.), an isotonic (sodium chloride, glycerin and sugar alcohol such asmannitol etc.), a pH adjusting agent (hydrochloric acid, citric acid,sodium hydroxide etc.) and a suspending agent (methylcellulose etc.) canbe used and, when formulated into a suppository, for example, asuppository base (cacao butter, macrogol etc.), and the like can beappropriately selected and used.

Examples of a preparation which is orally administered include powders,granules, tablets, capsules, syrups, solutions, aerosols and the like.When a preparation is formulated into a powder, granules, a tablet andthe like, any pharmaceutical carriers which are suitable for formulatinga solid preparation, for example, an excipient (starch, glucose,fructose, white sugar etc.), a lubricant (magnesium stearate etc.), adisintegrating agent (starch, crystalline cellulose etc.), a binder(starch, gum arabic etc.) can be used, and a preparation may be coatedwith a coating agent (gelatin, white sugar etc.). In addition, when apreparation is formulated into a syrup or a solution, for example, astabilizer (sodium edetate etc.), a suspending agent (gum arabic,carmerose etc.), a corrigent (simple syrup, glucose etc.) and aflavoring agent can be appropriately selected and used.

Examples of a topical preparation include ointments, creams, lotions,nose drops and topical ocular agents, preferably topical ocular agents.Examples of a topical ocular agent include eye drops, ocular ointmentsand sustained-release preparations, more preferably eye drops. In thesetopical preparations, in addition to the VIP derivatives of the presentinvention, for example, known compounds such as an ointment base(vaseline, lanolin etc.), a solvent (physiological saline, purifiedwater etc.), a stabilizer (sodium edetate, citric acid etc.), a wettingagent (glycerin etc.), an emulsifier (polyvinylpyrrolidone etc.), asuspending agent (hydroxypropylmethylcellulose, hydroxymethylcellulose,methylcellulose etc.), a surfactant (Polysorbate 80, polyoxyethylenehydrogenated castor oil etc.), a preservative (benzalkonium chloride,parabens, chlorobutanol etc.), a buffer (boric acid, borax, sodiumacetate, citrate buffer, phosphate buffer etc.), an isotonic (sodiumchloride, glycerin, mannitol etc.) and a pH adjusting agent(hydrochloric acid, sodium hydroxide etc.) can be appropriately selectedand used.

In addition, as an topical ocular sustained-release preparation, moldedgels such as collagen and the like, intraocular implants and scleraplugs obtained by molding a biodegradable polymer such as polylacticacid, or non-biodegradable intraocular implants can be used.

Generally, for the purpose of preventing peptides from adsorbing onto aglass or resin container, an adsorption preventing component can beutilized. The adsorption preventing component utilized herein is acompound which hydrophobically binds to a wall surface of a storagecontainer to prevent adhesion, more specifically, a compound which has ahydrophobic group in a molecule and has the surface active action, andan anion-charged protein. Examples of the former include polyoxyethylenealcohol ether, polyoxyethylene fatty acid ester, polyoxyethylenehydrogenated castor oil, glycerin fatty acid ester, sorbitan fatty acidester, polyoxyethylene sorbitan fatty acid ester and the like, andexamples of the latter anion-charged protein include gelatin, albumin,polygenin and the like. Examples of gelatin, are not limited to, butinclude gelatin purified according to the Japanese Pharmacopoeia and thelike. Gelatins as an adsorption preventing component may be used aloneor in combination of two or more thereof. As albumin, there are albuminshaving no antigenicity to a human, and the concentration of albumin tobe incorporated is usually about 0.01 to 50 w/v %, preferably about 0.1to 2.0 w/v %. As a solvent for dissolving an adsorption preventingcomponent and peptides, any solvents can be used as long as they arephysiologically acceptable as a solvent for injection, and preferredexamples thereof include water for injection according to the JapanesePharmacopoeia and physiological saline. Alternatively, the effect ofadsorption prevention can be obtained by coating silicone on an internalwall of a container.

By administering the dry eye remedy of the present invention to awarm-blooded animal (mammal such as rat, rabbit, cat, dog, pig, monkeyand human, birds such as pigeon and chicken and turkey), lacrimalsecretion is promoted. When the dry eye remedy of the present inventionis administered to an adult patient, a dose per once is usually 0.00001to 100 mg, preferably 0.0001 to 0.1 mg in case of an injectablepreparation, and is usually 0.1 to 500 mg, preferably 1 to 20 mg in caseof oral administration, in terms of the VIP derivative. When the remedyis topically applied to eyes of an adult patient, usually, eye dropscontaining the VIP derivative at 0.001 to 3.0 w/v %, preferably 0.01 to0.5 w/v % are applied to eyes at 20 to 50 μl per one application, onceto eight times per day.

The dry eye remedy of the present invention can contain an appropriatecombination of the VIP derivative and other ingredients for treating dryeye depending on the purpose and the necessity. In addition, as long asit is not contrary to the object of the present invention, the remedycan be used by combining with other pharmacologically activeingredients.

The dry eye remedy of the present invention can be used for treating dryeye such as decreased lacrimation, ocular xerosis, Sjögren's syndrome,keratoconjunctivitis sicca, Stevens-Johnson syndrome, dry eye associatedwith VDT (Visual Display Terminal) work and the like. Further, thepresent remedy is also useful as a remedy for diseases associated withdry eye such as corneal and conjuntival epithlial disorder, cornealepithelial erosion, corneal ulcer, limbi palpebrales imflammatory,ocular pemphigus, vernal conjunctivitis, allergic conjunctivitis, andthe like.

EXAMPLES

Hereinafter, the present invention will be illustrated in more detail byway of Synthesis Examples, Preparation Examples and Test Examples, butthe present invention is not limited to them.

The meanings of abbreviations used in the following Synthesis Examplesare as follows:

MBHA: p-methylbenzhydrylamine

MeOH: methanol

Boc: t-buthoxycarbonyl group

TFA: trifluoroacetic acid

TEA: triethylamine

Cl₂-Bzl: dichlorobenzyl group

Cl-Z: chlorobenzyloxycarbonyl group

Xan: xanthyl group

Tos: p-toluenesulfonyl group

Bzl: benzyl group

OcHex: O-cyclohexyl group

Bom: benzyloxymethyl group

DCC: dicyclohexylcarbodiimide

Synthesis Example 1 Preparation of Peptide 5

Peptide 5 having an amino acid sequence shown in SEQ ID NO: 5 wasprepared according to a conventional method of peptide solid phasesynthesis.

A MBHA resin HCl salt (polystyrene-1% divinylbenzene copolymer, 100 to200 mesh) was added to a manual synthesis reaction tank (made of glass,φ6.0×29.5 cm), which was washed with a 2 to 3-fold volume of the resinof MeOH while stirring and, then, washed with CH₂Cl₂ (2 to 3-fold volumeof the resin) while stirring, to swell the resin. A neutralizationreaction was performed with 10% triethylamine/CH₂Cl₂, and DCC andN-hydroxybenzotriazole were added to perform a condensation reactionusing Boc-Arg(Tos)-OH corresponding to a C-terminal amino acid in anamount of about 2-fold equivalent that of the resin. After the reactionfor about 2 hours (with stirring), the reaction mixture was washed withMeOH and CH₂Cl₂ and, after confirmation of disappearance of the α-aminogroup by Kaiser test, deprotection was performed by treatment with 50%TFA/CH₂Cl₂ for 30 minutes. Then, the reaction mixture was neutralizedwith 10% TEA/CH₂Cl₂, washed again with MeOH and CH₂Cl₂, and Kaiser testwas performed again to confirm the deprotecting reaction. Afterconfirmation, in order to perform coupling of 2^(nd) Boc-Arg(Tos)-OHfrom C-terminal, the similar step was repeated. Thereafter,coupling/deprotection was performed successively in an order ofBoc-Gly-OH, Boc-Asn(Xan)-OH, Boc-Leu-OH, Boc-Ile-OH, Boc-Ser(Bzl)-OH,Boc-Asn(Xan)-OH, Boc-Leu-OH, Boc-Tyr(Cl₂-Bzl)-OH, Boc-Arg(Tos)-OH,Boc-Arg(Tos)-OH, Boc-Val-OH, Boc-Ala-OH, Boc-Leu-OH, Boc-Gln(Xan)-OH,Boc-Arg(Tos)-OH, Boc-Arg (Tos)-OH, Boc-Leu-OH, Boc-Arg (Tos)-OH,Boc-Thr(Bzl)-OH, Boc-Tyr(Cl₂-Bzl)-OH, Boc-Asn(Xan)-OH,Boc-Asp(OcHex)-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH, Boc-Val-OH, Boc-Ala-OH,Boc-Asp(OcHex)-OH, Boc-Ser(Bzl)-OH, to obtain a protected peptide resincorresponding to Peptide 5;His(Bom)-Ser(Bzl)-Asp(OcHex)-Ala-Val-Phe-Thr(SEQ ID NO: 45) (Bzl)-Asp(OcHex)-Asn-Tyr (Cl₂-Bzl)-Thr (Bzl)-Arg(Tos)-Leu-Arg(Tos)-Arg(Tos)-Gln-Leu-Ala-Val-Arg(SEQ ID NO: 46)(Tos)-Arg(Tos)-Tyr(Cl₂-Bzl)-Leu-Asn-Ser(Bzl)-Ile-Leu-Asn-Gly-Arg (SEQ IDNO: 47) (Tos)-Arg(Tos)-MBHA.

To the resulting protected peptide-MBHA resin was added 100 mL ofanhydrous hydrogen fluoride in the presence of anisole to react them.After the reaction, anhydrous hydrogen fluoride was distilled off underreduced pressure, the residue was washed with ether, and 10% acetic acidwas added thereto to extract the peptide. The extract was purified byreverse phase column chromatography (stepwise gradient between 10%acetonitrile and 50% acetonitrile), followed by lyophilizing to obtainPeptide 5.

Synthesis Example 2 Preparation of Peptide 15

Peptide 15 having an amino acid sequence shown in SEQ ID NO: 15 wassynthesized in the same manner as that of Peptide 5. Namely,coupling/deprotection was performed successively on a MBHA resin in anorder of Boc-Leu-OH, Boc-Tyr(Cl₂-Bzl)-OH, Boc-Lys(Cl-Z)-OH,Boc-Lys(Cl-Z)-OH, Boc-Val-OH, Boc-Ala-OH, Boc-Leu-OH, Boc-Gln(Xan)-OH,Boc-Lys(Cl-Z)-OH, Boc-Arg(Tos)-OH, Boc-Leu-OH, Boc-Arg(Tos)-OH,Boc-Thr(Bzl)-OH, Boc-Tyr(Cl₂-Bzl)-OH, Boc-Asn(Xan)-OH,Boc-Asp(OcHex)-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH, Boc-Val-OH, Boc-Ala-OH,Boc-Asp(OcHex)-OH, Boc-Ser(Bzl)-OH, and Boc-His (Bom)-OH, to obtain aprotected peptide resin corresponding to Peptide 15;His(Bom)-Ser(Bzl)-Asp(OcHex)-Ala-Val-Phe-Thr (SEQ ID NO: 45)(Bzl)-Asp(OcHex)-Asn-Tyr(Cl₂-Bzl)-Thr(Bzl)-Arg(Tos)-Leu-Arg(Tos)-Lys(Cl-Z)-Gln-Leu-Ala-Val-Lys(SEQID NO: 48) (Cl-Z)-Lys(Cl-Z)-Tyr(Cl₂-Bzl)-Leu-MBHA.

To the resulting protected peptide-MBHA resin was added 100 mL ofanhydrous hydrogen fluoride in the presence of anisole to react them.After the reaction, anhydrous hydrogen fluoride was distilled off underreduced pressure, the residue was washed with ether, and 10% acetic acidwas added thereto to extract the peptide. The extract was purified byreverse phase column chromatography (stepwise gradient between 10%acetonitrile and 50% acetonitrile), followed by lyophilizing to obtainPeptide 15.

Synthesis Example 3 Preparation of Peptide 16

Peptide 16 having an amino acid sequence shown in SEQ ID NO: 16 wassynthesized in the same manner as that of Peptide 5. Namely,coupling/deprotection was performed on a MBHA resin successively in anorder of Boc-Leu-OH, Boc-Ile-OH, Boc-Ser(Bzl)-OH, Boc-Asn(Xan)-OH,Boc-Leu-OH, Boc-Tyr(Cl₂-Bzl)-OH, Boc-Arg(Tos)-OH, Boc-Arg(Tos)-OH,Boc-Val-OH, Boc-Ala-OH, Boc-Met-OH, Boc-Gln(Xan)-OH, Boc-Arg(Tos)-OH,Boc-Arg(Tos)-OH, Boc-Leu-OH, Boc-Arg(Tos)-OH, Boc-Thr(Bzl)-OH,Boc-Tyr(Cl₂-Bzl)-OH, Boc-Asn(Xan)-OH, Boc-Asp(OcHex)-OH,Boc-Thr(Bzl)-OH, Boc-Phe-OH, Boc-Val-OH, Boc-Ala-OH, Boc-Asp(OcHex)-OH,Boc-Ser(Bzl)-OH, and Boc-His(Bom)-OH, to obtain a protected peptideresin corresponding to Peptide 16;His(Bom)-Ser(Bzl)-Asp(OcHex)-Ala-Val-Phe-Thr(SEQ ID NO: 45)(Bzl)-Asp(OcHex)-Asn-Tyr(Cl₂-Bzl)-Thr(Bzl)-Arg(Tos)-Leu-Arg(Tos)-Arg(Tos)-Gln-Met-Ala-Val-Arg(SEQID NO: 49) (Tos)-Arg(Tos)-Tyr(Cl₂-Bzl)-Leu-Asn-Ser(Bzl)-Ile-Leu-MBHA.

To the resulting protected peptide-MBHA resin was added 100 mL ofanhydrous hydrogen fluoride in the presence of anisole to react them.After the reaction, anhydrous hydrogen fluoride was distilled off underreduced pressure, the residue was washed with ether, and 10% acetic acidwas added thereto to extract the peptide. The extract was purified byreverse phase column chromatography (stepwise gradient between 10%acetonitrile and 50% acetonitrile), followed by lyophilizing to obtainPeptide 16.

Synthesis Example 4 Preparation of Peptide 17

Peptide 17 having an amino acid sequence shown in SEQ ID NO: 17 wassynthesized in the same manner as that of Peptide 5. Namely,coupling/deprotection was performed on a MBHA resin successively in anorder of Boc-Ile-OH, Boc-Ser(Bzl)-OH, Boc-Asn(Xan)-OH, Boc-Leu-OH,Boc-Tyr(Cl₂-Bzl)-OH, Boc-Arg(Tos)-OH, Boc-Arg(Tos)-OH, Boc-Val-OH,Boc-Ala-OH, Boc-Met-OH, Boc-Gln(Xan)-OH, Boc-Arg(Tos)-OH,Boc-Arg(Tos)-OH, Boc-Leu-OH, Boc-Arg(Tos)-OH, Boc-Thr(Bzl)-OH,Boc-Tyr(Cl₂-Bzl)-OH, Boc-Asn(Xan)-OH, Boc-Asp(OcHex)-OH,Boc-Thr(Bzl)-OH, Boc-Phe-OH, Boc-Val-OH, Boc-Ala-OH, Boc-Asp(OcHex)-OH,Boc-Ser(Bzl)-OH, and Boc-His(Bom)-OH, to obtain a protected peptideresin corresponding to Peptide 17;His(Bom)-Ser(Bzl)-Asp(OcHex)-Ala-Val-Phe-Thr(SEQ ID NO: 45)(Bzl)-Asp(OcHex)-Asn-Tyr(Cl₂-Bzl)-Thr(Bzl)-Arg(Tos)-Leu-Arg(Tos)-Arg(Tos)-Gln-Met-Ala-Val-Arg(SEQID NO: 49) (Tos)-Arg(Tos)-Tyr(Cl₂-Bzl)-Leu-Asn-Ser(Bzl)-Ile-MBHA.

To the resulting protected peptide-MBHA resin was added 100 mL ofanhydrous hydrogen fluoride in the presence of anisole to react them.After the reaction, anhydrous hydrogen fluoride was distilled off underreduced pressure, the residue was washed with ether, and 10% acetic acidwas added thereto to extract the peptide. The extract was purified byreverse phase column chromatography (stepwise gradient between 10%acetonitrile and 50% acetonitrile), followed by lyophilizing to obtainPeptide 17.

Synthesis Example 5 Preparation of Peptide 18

Peptide 18 having an amino acid sequence shown in SEQ ID NO:18 wassynthesized in the same manner as that of Peptide 5. Namely,coupling/deprotection was performed on a MBHA resin successively in anorder of Boc-Ser(Bzl)-OH, Boc-Asn(Xan)-OH, Boc-Leu-OH,Boc-Tyr(Cl₂-Bzl)-OH, Boc-Arg(Tos)-OH, Boc-Arg(Tos)-OH, Boc-Val-OH,Boc-Ala-OH, Boc-Met-OH, Boc-Gln(Xan)-OH, Boc-Arg(Tos)-OH,Boc-Arg(Tos)-OH, Boc-Leu-OH, Boc-Arg (Tos)-OH, Boc-Thr(Bzl)-OH, Boc-Tyr(Cl₂-Bzl)-OH, Boc-Asn(Xan)-OH, Boc-Asp(OcHex)-OH, Boc-Thr(Bzl)-OH,Boc-Phe-OH, Boc-Val-OH, Boc-Ala-OH, Boc-Asp(OcHex)-OH, Boc-Ser(Bzl)-OH,and Boc-His(Bom)-OH, to obtain a protected peptide resin correspondingto Peptide 18; His(Bom)-Ser(Bzl)-Asp(OcHex)-Ala-Val-Phe-Thr(SEQ ID NO:45) (Bzl)-Asp (OcHex)-Asn-Tyr (Cl₂-Bzl)-Thr (Bzl)-Arg (Tos)-Leu-Arg(Tos)-Arg(Tos)-Gln-Met-Ala-Val-Arg(SEQ ID NO: 49) (Tos)-Arg(Tos)-Tyr(Cl₂-Bzl)-Leu-Asn-Ser (Bzl)-MBHA.

To the resulting protected peptide-MBHA resin was added 100 mL ofanhydrous hydrogen fluoride in the presence of anisole to react them.After the reaction, anhydrous hydrogen fluoride was distilled off underreduced pressure, the residue was washed with ether, and 10% acetic acidwas added thereto to extract the peptide. The extract was purified byreverse phase column chromatography (stepwise gradient between 10%acetonitrile and 50% acetonitrile), followed by lyophilizing to obtainPeptide 18.

Synthesis Example 6 Preparation of Peptide 19

Peptide 19 having an amino acid sequence shown in SEQ ID NO:19 wassynthesized in the same manner as that of Peptide 5. Namely,coupling/deprotection was performed on a MBHA resin successively in anorder of Boc-Asn(Xan)-OH, Boc-Leu-OH, Boc-Tyr (Cl₂-Bzl)-OH, Boc-Arg(Tos)-OH, Boc-Arg(Tos)-OH, Boc-Val-OH, Boc-Ala-OH, Boc-Met-OH,Boc-Gln(Xan)-OH, Boc-Arg(Tos)-OH, Boc-Arg(Tos)-OH, Boc-Leu-OH,Boc-Arg(Tos)-OH, Boc-Thr(Bzl)-OH, Boc-Tyr(Cl₂-Bzl)-OH, Boc-Asn(Xan)-OH,Boc-Asp(OcHex)-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH, Boc-Val-OH, Boc-Ala-OH,Boc-Asp(OcHex)-OH, Boc-Ser(Bzl)-OH, and Boc-His(Bom)-OH, to obtain aprotected peptide resin corresponding to Peptide 19;His(Bom)-Ser(Bzl)-Asp(OcHex)-Ala-Val-Phe-Thr(SEQ ID NO: 45)(Bzl)-Asp(OcHex)-Asn-Tyr(Cl₂-Bzl)-Thr(Bzl)-Arg(Tos)-Leu-Arg(Tos)-Arg(Tos)-Gln-Met-Ala-Val-Arg(SEQID NO: 49) (Tos)-Arg(Tos)-Tyr(Cl₂-Bzl)-Leu-Asn-MBHA.

To the resulting protected peptide-MBHA resin was added 100 mL ofanhydrous hydrogen fluoride in the presence of anisole to react them.After the reaction, anhydrous hydrogen fluoride was distilled off underreduced pressure, the residue was washed with ether, and 10% acetic acidwas added thereto to extract the peptide. The extract was purified byreverse phase column chromatography (stepwise gradient between 10%acetonitrile and 50% acetonitrile), followed by lyophilizing to obtainPeptide 19.

Synthesis Example 7 Preparation of Peptide 20

Peptide 20 having an amino acid sequence shown in SEQ ID NO: 20 wassynthesized in the same manner as that of Peptide 5. Namely,coupling/deprotection was performed on a MBHA resin successively in anorder of Boc-Leu-OH, Boc-Tyr(Cl₂-Bzl)-OH, Boc-Arg(Tos)-OH,Boc-Arg(Tos)-OH, Boc-Val-OH, Boc-Ala-OH, Boc-Met-OH, Boc-Gln(Xan)-OH,Boc-Arg(Tos)-OH, Boc-Arg(Tos)-OH, Boc-Leu-OH, Boc-Arg(Tos)-OH,Boc-Thr(Bzl)-OH, Boc-Tyr(Cl₂-Bzl)-OH, Boc-Asn(Xan)-OH,Boc-Asp(OcHex)-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH, Boc-Val-OH, Boc-Ala-OH,Boc-Asp(OcHex)-OH, Boc-Ser(Bzl)-OH, and Boc-His(Bom)-OH, to obtain aprotected peptide resin corresponding to Peptide 20;His(Bom)-Ser(Bzl)-Asp(OcHex)-Ala-Val-Phe-Thr(SEQ ID NO: 45)(Bzl)-Asp(OcHex)-Asn-Tyr(Cl₂-Bzl)-Thr(Bzl)-Arg(Tos)-Leu-Arg(Tos)-Arg(Tos)-Gln-Met-Ala-Val-Arg(SEQID NO: 49) (Tos)-Arg(Tos)-Tyr(Cl₂-Bzl)-Leu-MBHA.

To the resulting protected peptide-MBHA resin was added 100 mL ofanhydrous hydrogen fluoride in the presence of anisole to react them.After the reaction, anhydrous hydrogen fluoride was distilled off underreduced pressure, the residue was washed with ether, and 10% acetic acidwas added thereto to extract the peptide. The extract was purified byreverse phase column chromatography (stepwise gradient between 10%acetonitrile and 50% acetonitrile), followed by lyophilizing to obtainPeptide 20.

Synthesis Example 8 Preparation of Peptide 21

Peptide 21 having an amino acid sequence shown in SEQ ID NO: 21 wassynthesized in the same manner as that of Peptide 5. Namely,coupling/deprotection was performed on a MBHA resin successively in anorder of Boc-Leu-OH, Boc-Ile-OH, Boc-Ser(Bzl)-OH, Boc-Asn(Xan)-OH,Boc-Leu-OH, Boc-Tyr(Cl₂-Bzl)-OH, Boc-Arg(Tos)-OH, Boc-Arg(Tos)-OH,Boc-Val-OH, Boc-Ala-OH, Boc-Leu-OH, Boc-Gln(Xan)-OH, Boc-Arg(Tos)-OH,Boc-Arg(Tos)-OH, Boc-Leu-OH, Boc-Arg(Tos)-OH, Boc-Thr(Bzl)-OH,Boc-Tyr(Cl₂-Bzl)-OH, Boc-Asn(Xan)-OH, Boc-Asp(OcHex)-OH,Boc-Thr(Bzl)-OH, Boc-Phe-OH, Boc-Val-OH, Boc-Ala-OH, Boc-Asp(OcHex)-OH,Boc-Ser(Bzl)-OH, and Boc-His(Bom)-OH, to obtain a protected peptideresin corresponding to Peptide 21;His(Bom)-Ser(Bzl)-Asp(OcHex)-Ala-Val-Phe-Thr(SEQ ID NO: 45)(Bzl)-Asp(OcHex)-Asn-Tyr(Cl₂-Bzl)-Thr(Bzl)-Arg(Tos)-Leu-Arg(Tos)-Arg(Tos)-Gln-Leu-Ala-Val-Arg(SEQID NO: 46) (Tos)-Arg(Tos)-Tyr(Cl₂-Bzl)-Leu-Asn-Ser(Bzl)-Ile-Leu-MBHA.

To the resulting protected peptide-MBHA resin was added 100 mL ofanhydrous hydrogen fluoride in the presence of anisole to react them.After the reaction, anhydrous hydrogen fluoride was distilled off underreduced pressure, the residue was washed with ether, and 10% acetic acidwas added thereto to extract the peptide. The extract was purified byreverse phase column chromatography (stepwise gradient between 10%acetonitrile and 50% acetonitrile), followed by lyophilizing to obtainPeptide 21.

Synthesis Example 9 Preparation of Peptide 22

Peptide 22 having an amino acid sequence shown in SEQ ID NO: 22 wassynthesized in the same manner as that of Peptide 5. Namely,coupling/deprotection was performed on a MBHA resin successively in anorder of Boc-Ile-OH, Boc-Ser(Bzl)-OH, Boc-Asn(Xan)-OH, Boc-Leu-OH,Boc-Tyr(Cl₂-Bzl)-OH, Boc-Arg(Tos)-OH, Boc-Arg(Tos)-OH, Boc-Val-OH,Boc-Ala-OH, Boc-Leu-OH, Boc-Gln(Xan)-OH, Boc-Arg(Tos)-OH,Boc-Arg(Tos)-OH, Boc-Leu-OH, Boc-Arg(Tos)-OH, Boc-Thr(Bzl)-OH,Boc-Tyr(Cl₂-Bzl)-OH, Boc-Asn(Xan)-OH, Boc-Asp(OcHex)-OH,Boc-Thr(Bzl)-OH, Boc-Phe-OH, Boc-Val-OH, Boc-Ala-OH, Boc-Asp(OcHex)-OH,Boc-Ser(Bzl)-OH and Boc-His(Bom)-OH, to obtain a protected peptide resincorresponding to Peptide 22;His(Bom)-Ser(Bzl)-Asp(OcHex)-Ala-Val-Phe-Thr(SEQ ID NO: 45)(Bzl)-Asp(OcHex)-Asn-Tyr(Cl₂-Bzl)-Thr(Bzl)-Arg(Tos)-Leu-Arg(Tos)-Arg(Tos)-Gln-Leu-Ala-Val-Arg(SEQID NO: 46) (Tos)-Arg(Tos)-Tyr(Cl₂-Bzl)-Leu-Asn-Ser(Bzl)-Ile-MBHA.

To the resulting protected peptide-MBHA resin was added 100 mL ofanhydrous hydrogen fluoride in the presence of anisole to react them.After the reaction, anhydrous hydrogen fluoride was distilled off underreduced pressure, the residue was washed with ether, and 10% acetic acidwas added thereto to extract the peptide. The extract was purified byreverse phase column chromatography (stepwise gradient between 10%acetonitrile and 50% acetonitrile), followed by lyophilizing to obtainPeptide 22.

Synthesis Example 10 Preparation of Peptide 23

Peptide 23 having an amino acid sequence shown in SEQ ID NO: 23 wassynthesized in the same manner as that of Peptide 5. Namely,coupling/deprotection was performed on a MBHA resin successively in anorder of Boc-Ser(Bzl)-OH, Boc-Asn(Xan)-OH, Boc-Leu-OH, Boc-Tyr(Cl₂-Bzl)-OH, Boc-Arg(Tos)-OH, Boc-Arg(Tos)-OH, Boc-Val-OH, Boc-Ala-OH,Boc-Leu-OH, Boc-Gln(Xan)-OH, Boc-Arg(Tos)-OH, Boc-Arg(Tos)-OH,Boc-Leu-OH, Boc-Arg(Tos)-OH, Boc-Thr(Bzl)-OH, Boc-Tyr(Cl₂-Bzl)-OH,Boc-Asn(Xan)-OH, Boc-Asp(OcHex)-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH,Boc-Val-OH, Boc-Ala-OH, Boc-Asp(OcHex)-OH, Boc-Ser(Bzl)-OH, andBoc-His(Bom)-OH, to obtain a protected resin corresponding to Peptide23; His(Bom)-Ser(Bzl)-Asp(OcHex)-Ala-Val-Phe-Thr(SEQ ID NO: 45)(Bzl)-Asp(OcHex)-Asn-Tyr (Cl₂-Bzl)-Thr (Bzl)-Arg (Tos)-Leu-Arg (Tos)-Arg(Tos)-Gln-Leu-Ala-Val-Arg(SEQ ID NO: 46) (Tos)-Arg(Tos)-Tyr(Cl₂-Bzl)-Leu-Asn-Ser(Bzl)-MBHA.

To the resulting protected peptide-MBHA resin was added 100 mL ofanhydrous hydrogen fluoride in the presence of anisole to react them.After the reaction, anhydrous hydrogen fluoride was distilled off underreduced pressure, the residue was washed with ether, and 10% acetic acidwas added thereto to extract the peptide. The extract was purified byreverse phase column chromatography (stepwise gradient between 10%acetonitrile and 50% acetonitrile), followed by lyophilizing to obtainPeptide 23.

Synthesis Example 11 Preparation of Peptide 24

Peptide 24 having an amino acid sequence shown in SEQ ID NO: 24 wassynthesized in the same manner as that of Peptide 5. Namely,coupling/deprotection was performed on a MBHA resin successively in anorder of Boc-Asn(Xan)-OH, Boc-Leu-OH, Boc-Tyr (Cl₂-Bzl)-OH,Boc-Arg(Tos)-OH, Boc-Arg(Tos)-OH, Boc-Val-OH, Boc-Ala-OH, Boc-Leu-OH,Boc-Gln (Xan)-OH, Boc-Arg (Tos)-OH, Boc-Arg (Tos)-OH, Boc-Leu-OH,Boc-Arg(Tos)-OH, Boc-Thr(Bzl)-OH, Boc-Tyr(Cl₂-Bzl)-OH, Boc-Asn(Xan)-OH,Boc-Asp(OcHex)-OH, Boc-Thr(Bzl)-OH Boc-Phe-OH, Boc-Val-OH, Boc-Ala-OH,Boc-Asp(OcHex)-OH, Boc-Ser(Bzl)-OH, and Boc-His(Bom)-OH, to obtain aprotected peptide resin corresponding to Peptide 24;His(Bom)-Ser(Bzl)-Asp(OcHex)-Ala-Val-Phe-Thr(SEQ ID NO: 45)(Bzl)-Asp(OcHex)-Asn-Tyr(Cl₂-Bzl)-Thr(Bzl)-Arg(Tos)-Leu-Arg(Tos)-Arg(Tos)-Gln-Leu-Ala-Val-Arg(SEQID NO: 46) (Tos)-Arg(Tos)-Tyr(Cl₂-Bzl)-Leu-Asn-MBHA.

To the resulting protected peptide-MBHA resin was added 100 mL ofanhydrous hydrogen fluoride in the presence of anisole to react them.After the reaction, anhydrous hydrogen fluoride was distilled off underreduced pressure, the residue was washed with ether, and 10% acetic acidwas added thereto to extract the peptide. The extract was purified byreverse phase column chromatography (stepwise gradient between 10%acetonitrile and 50% acetonitrile), followed by lyophilizing to obtainPeptide 24.

Synthesis Example 12 Preparation of Peptide 25

Peptide 25 having an amino acid sequence shown in SEQ ID NO: 25 wassynthesized in the same manner as that of Peptide 5. Namely,coupling/deprotection was performed on a MBHA resin successively in anorder of Boc-Leu-OH, Boc-Tyr(Cl₂-Bzl)-OH, Boc-Arg(Tos)-OH,Boc-Arg(Tos)-OH, Boc-Val-OH, Boc-Ala-OH, Boc-Leu-OH, Boc-Gln(Xan)-OH,Boc-Arg(Tos)-OH, Boc-Arg(Tos)-OH, Boc-Leu-OH, Boc-Arg(Tos)-OH, Boc-Thr(Bzl)-OH, Boc-Tyr(Cl₂-Bzl)-OH, Boc-Asn(Xan)-OH, Boc-Asp(OcHex)-OH,Boc-Thr(Bzl)-OH, Boc-Phe-OH, Boc-Val-OH, Boc-Ala-OH, Boc-Asp(OcHex)-OH,Boc-Ser(Bzl)-OH, and Boc-His(Bom)-OH, to obtain a protected peptideresin corresponding to Peptide 25;His(Bom)-Ser(Bzl)-Asp(OcHex)-Ala-Val-Phe-Thr(SEQ ID NO: 45)(Bzl)-Asp(OcHex)-Asn-Tyr(Cl₂-Bzl)-Thr(Bzl)-Arg(Tos)-Leu-Arg(Tos)-Arg(Tos)-Gln-Leu-Ala-Val-Arg(SEQ ID NO: 46) (Tos)-Arg (Tos)-Tyr (Cl₂-Bzl)-Leu-MBHA.

To the resulting protected peptide-MBHA resin was added 100 mL ofanhydrous hydrogen fluoride in the presence of anisole to react them.After the reaction, anhydrous hydrogen fluoride was distilled off underreduced pressure, the residue was washed with ether, and 10% acetic acidwas added thereto to extract the peptide. The extract was purified byreverse phase column chromatography (stepwise gradient between 10%acetonitrile and 50% acetonitrile), followed by lyophilizing to obtainPeptide 25.

Synthesis Example 13 Preparation of Peptide 1

Peptide 1 having an amino acid sequence shown in SEQ ID NO: 1 wassynthesized in the same manner as that of Peptide 5. Namely,coupling/deprotection was performed on a MBHA resin successively in anorder of Boc-Gly-OH, Boc-Asn(Xan)-OH, Boc-Leu-OH, Boc-Ile-OH,Boc-Ser(Bzl)-OH, Boc-Asn(Xan)-OH, Boc-Leu-OH, Boc-Tyr(Cl₂-Z)-OH,Boc-Arg(Tos)-OH, Boc-Arg(Tos)-OH, Boc-Val-OH, Boc-Ala-OH, Boc-Leu-OH,Boc-Gln(Xan)-OH, Boc-Arg(Tos)-OH, Boc-Arg(Tos)-OH, Boc-Leu-OH,Boc-Arg(Tos)-OH, Boc-Thr(Bzl)-OH, Boc-Tyr(Cl₂-Z)-OH, Boc-Asn(Xan)-OH,Boc-Asp(OcHex)-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH, Boc-Val-OH, Boc-Ala-OH,Boc-Asp(OcHex)-OH, Boc-Ser(Bzl)-OH, and Boc-His(Bom)-OH, to obtain aprotected peptide resin corresponding to Peptide 1;His(Bom)-Ser(Bzl)-Asp(OcHex)-Ala-Val-Phe-Thr(SEQ ID NO: 45)(Bzl)-Asp(OcHex)-Asn-Tyr(Cl₂-Bzl)-Thr(Bzl)-Arg(Tos)-Leu-Arg(Tos)-Arg(Tos)-Gln-Leu-Ala-Val-Arg(SEQ ID NO: 46) (Tos)-Arg (Tos)-Tyr(Cl₂-Bzl)-Leu-Asn-Ser(Bzl)-Ile-Leu-Asn-Gly-MBHA. To the resultingprotected peptide-MBHA resin was added anhydrous hydrogen fluoride inthe presence of anisole to react them. After the reaction, anhydroushydrogen fluoride was distilled off under reduced pressure, the residuewas washed with ether, and 10% acetic acid was added thereto to extractthe peptide. The extract was purified by reverse phase columnchromatography, and lyophilized to obtain Peptide 1.

Synthesis Example 14 Preparation of Peptide 2

Peptide 2 having an amino acid sequence shown in SEQ ID NO: 2 wassynthesized as in the process of preparing Peptide 5. Namely,coupling/deprotection was performed on a MBHA resin successively in anorder of Boc-Lys(Cl-Z)-OH, Boc-Gly-OH, Boc-Asn(Xan)-OH, Boc-Leu-OH,Boc-Ile-OH, Boc-Ser(Bzl)-OH, Boc-Asn(Xan)-OH, Boc-Leu-OH,Boc-Tyr(Cl₂-Z)-OH, Boc-Arg(Tos)-OH, Boc-Arg(Tos)-OH, Boc-Val-OH,Boc-Ala-OH, Boc-Leu-OH, Boc-Gln(Xan)-OH, Boc-Arg(Tos)-OH,Boc-Arg(Tos)-OH, Boc-Leu-OH, Boc-Arg(Tos)-OH, Boc-Thr(Bzl)-OH,Boc-Tyr(Cl₂-Z)-OH, Boc-Asn(Xan)-OH, Boc-Asp(OcHex)-OH, Boc-Thr(Bzl)-OH,Boc-Phe-OH, Boc-Val-OH, Boc-Ala-OH, Boc-Asp(OcHex)-OH, Boc-Ser(Bzl)-OH,and Boc-His(Bom)-OH, to obtain a protected peptide resin correspondingto Peptide 2; His(Bom)-Ser(Bzl)-Asp(OcHex)-Ala-Val-Phe-Thr (SEQ ID NO:45)(Bzl)-Asp(OcHex)-Asn-Tyr(Cl₂-Bzl)-Thr(Bzl)-Arg(Tos)-Leu-Arg(Tos)-Arg(Tos)-Gln-Leu-Ala-Val-Arg(SEQID NO: 46) (Tos)-Arg (Tos)-Tyr (Cl₂-Bzl)-Leu-Asn-Ser(Bzl)-Ile-Leu-Asn-Gly-Lys (SEQ ID NO: 50) (Cl-Z)-MBHA. To the resultingprotected peptide-MBHA resin was added anhydrous hydrogen fluoride inthe presence of anisole to react them. After the reaction, anhydroushydrogen fluoride was distilled off under reduced pressure, the residuewas washed with ether, and 10% acetic acid was added thereto to extractthe peptide. The extract was purified by reverse phase columnchromatography, and lyophilized to obtain Peptide 2.

Synthesis Example 15 Preparation of Peptide 3

Peptide 3 having an amino acid sequence shown in SEQ ID NO: 3 wassynthesized in the same manner as that of Peptide 5. Namely,coupling/deprotection was performed on a MBHA resin successively in anorder of Boc-Arg(Tos)-OH, Boc-Gly-OH, Boc-Asn(Xan)-OH, Boc-Leu-OH,Boc-Ile-OH, Boc-Ser(Bzl)-OH, Boc-Asn(Xan)-OH, Boc-Leu-OH,Boc-Tyr(Cl₂-Z)-OH, Boc-Arg(Tos)-OH, Boc-Arg(Tos)-OH, Boc-Val-OH,Boc-Ala-OH, Boc-Leu-OH, Boc-Gln(Xan)-OH, Boc-Arg(Tos)-OH,Boc-Arg(Tos)-OH, Boc-Leu-OH, Boc-Arg(Tos)-OH, Boc-Thr(Bzl)-OH,Boc-Tyr(Cl₂-Z)-OH, Boc-Asn(Xan)-OH, Boc-Asp(OcHex)-OH, Boc-Thr(Bzl)-OH,Boc-Phe-OH, Boc-Val-OH, Boc-Ala-OH, Boc-Asp(OcHex)-OH, Boc-Ser(Bzl)-OH,and Boc-His(Bom)-OH, to obtain a protected peptide resin correspondingto Peptide 3; His(Bom)-Ser(Bzl)-Asp(OcHex)-Ala-Val-Phe-Thr(SEQ ID NO:45)(Bzl)-Asp(OcHex)-Asn-Tyr(Cl₂-Bzl)-Thr(Bzl)-Arg(Tos)-Leu-Arg(Tos)-Arg(Tos)-Gln-Leu-Ala-Val-Arg(SEQID NO: 46)(Tos)-Arg(Tos)-Tyr(Cl₂-Bzl)-Leu-Asn-Ser(Bzl)-Ile-Leu-Asn-Gly-Arg (SEQ IDNO: 47) (Tos)-MBHA. To the resulting protected peptide-MBHA resin wasadded anhydrous hydrogen fluoride in the presence of anisole to reactthem. After the reaction, anhydrous hydrogen fluoride was distilled offunder reduced pressure, the residue was washed with ether, and 10%acetic acid was added thereto to extract the peptide. The extract waspurified by reverse phase column chromatography, and lyophilized toobtain Peptide 3.

Synthesis Example 16 Preparation of Peptide 4

Peptide 4 having an amino acid sequence shown in SEQ ID NO: 4 wassynthesized in the same manner as that of Peptide 5. Namely,coupling/deprotection was performed on a MBHA resin successively in anorder of Boc-Arg(Tos)-OH, Boc-Lys(Cl-Z)-OH, Boc-Gly-OH, Boc-Asn(Xan)-OH,Boc-Leu-OH, Boc-Ile-OH, Boc-Ser(Bzl)-OH, Boc-Asn(Xan)-OH, Boc-Leu-OH,Boc-Tyr (Cl₂-Z)-OH, Boc-Arg(Tos)-OH, Boc-Arg(Tos)-OH, Boc-Val-OH,Boc-Ala-OH, Boc-Leu-OH, Boc-Gln(Xan)-OH, Boc-Arg(Tos)-OH,Boc-Arg(Tos)-OH, Boc-Leu-OH, Boc-Arg(Tos)-OH, Boc-Thr(Bzl)-OH,Boc-Tyr(Cl₂-Z)-OH, Boc-Asn(Xan)-OH, Boc-Asp(OcHex)-OH, Boc-Thr(Bzl)-OH,Boc-Phe-OH, Boc-Val-OH, Boc-Ala-OH, Boc-Asp(OcHex)-OH, Boc-Ser(Bzl)-OH,and Boc-His(Bom)-OH, to obtain a protected peptide resin correspondingto Peptide 4; His(Bom)-Ser(Bzl)-Asp(OcHex)-Ala-Val-Phe-Thr(SEQ ID NO:45)(Bzl)-Asp(OcHex)-Asn-Tyr(Cl₂-Bzl)-Thr(Bzl)-Arg(Tos)-Leu-Arg(Tos)-Arg(Tos)-Gln-Leu-Ala-Val-Arg(SEQID NO: 46)(Tos)-Arg(Tos)-Tyr(Cl₂-Bzl)-Leu-Asn-Ser(Bzl)-Ile-Leu-Asn-Gly-Lys(SEQ IDNO: 50) (Cl-Z)-Arg(Tos)-MBHA. To the resulting protected peptide-MBHAresin was added anhydrous hydrogen fluoride in the presence of anisoleto react them. After the reaction, anhydrous hydrogen fluoride wasdistilled off under reduced pressure, the residue was washed with ether,and 10% acetic acid was added thereto to extract the peptide. Theextract was purified by reverse phase column chromatography, andlyophilized to obtain Peptide 4.

Synthesis Example 17 Preparation of Peptide 6

Peptide 6 having an amino acid sequence shown in SEQ ID NO: 6 wassynthesized in the same manner as that of Peptide 5. Namely,coupling/deprotection was performed on a PAM resin successively in anorder of Boc-Arg(Tos)-OH, Boc-Lys(Cl-Z)-OH, Boc-Gly-OH, Boc-Asn(Xan)-OH,Boc-Leu-OH, Boc-Ile-OH, Boc-Ser(Bzl)-OH, Boc-Asn(Xan)-OH, Boc-Leu-OH,Boc-Tyr(Cl₂-Z)-OH, Boc-Arg(Tos)-OH, Boc-Arg(Tos)-OH, Boc-Val-OH,Boc-Ala-OH, Boc-Leu-OH, Boc-Gln(Xan)-OH, Boc-Arg(Tos)-OH,Boc-Arg(Tos)-OH, Boc-Leu-OH, Boc-Arg(Tos)-OH, Boc-Thr(Bzl)-OH,Boc-Tyr(Cl₂-Z)-OH, Boc-Asn Xan)-OH, Boc-Asp(OcHex)-OH, Boc-Thr(Bzl)-OH,Boc-Phe-OH, Boc-Val-OH, Boc-Ala-OH, Boc-Asp(OcHex)-OH, Boc-Ser(Bzl)-OH,and Boc-His(Bom)-OH, to obtain a protected peptide resin correspondingto Peptide 6; His(Bom)-Ser(Bzl)-Asp(OcHex)-Ala-Val-Phe-Thr(SEQ ID NO:45) (Bzl)-Asp(OcHex)-Asn-Tyr(Cl₂-Bzl)-Thr(Bzl)-Arg(Tos)-Leu-Arg(Tos)-Arg(Tos)-Gln-Leu-Ala-Val-Arg(SEQ ID NO: 46)(Tos)-Arg(Tos)-Tyr(Cl₂-Bzl)-Leu-Asn-Ser(Bzl)-Ile-Leu-Asn-Gly-Lys(SEQ IDNO: 50) (Cl-Z)-Arg(Tos)-PAM. To the resulting protected peptide-PAMresin was added anhydrous hydrogen fluoride in the presence of anisoleto react them. After the reaction, anhydrous hydrogen fluoride wasdistilled off under reduced pressure, the residue was washed with ether,and 10% acetic acid was added thereto to extract the peptide. Theextract was purified by reverse phase column chromatography, andlyophilized to obtain Peptide 6.

Synthesis Example 18 Preparation of Peptide 7

Peptide 7 having an amino acid sequence shown in SEQ ID NO: 7 wassynthesized in the same manner as that of Peptide 5. Namely,coupling/deprotection was performed on a PAM resin successively in anorder of Boc-Asn(Xan)-OH, Boc-Leu-OH, Boc-Ile-OH, Boc-Ser(Bzl)-OH,Boc-Asn(Xan)-OH, Boc-Leu-OH, Boc-Tyr (Cl₂-Z)-OH, Boc-Lys(Cl-Z)-OH,Boc-Lys(Cl-Z)-OH, Boc-Val-OH, Boc-Ala-OH, Boc-Met-OH, Boc-Gln(Xan)-OH,Boc-Lys(Cl-Z)-OH, Boc-Arg(Tos)-OH, Boc-Leu-OH, Boc-Arg(Tos)-OH,Boc-Thr(Bzl)-OH, Boc-Tyr(Cl₂-Z)-OH, Boc-Asn(Xan)-OH, Boc-Asp(OcHex)-OH,Boc-Thr(Bzl)-OH, Boc-Phe-OH, Boc-Val-OH, Boc-Ala-OH, Boc-Asp(OcHex)-OH,Boc-Ser(Bzl)-OH, and Boc-His(Bom)-OH, to obtain a protected peptideresin corresponding to Peptide 7;His(Bom)-Ser(Bzl)-Asp(OcHex)-Ala-Val-Phe-Thr(SEQ ID NO: 45)(Bzl)-Asp(OcHex)-Asn-Tyr(Cl₂-Bzl)-Thr(Bzl)-Arg(Tos)-Leu-Arg(Tos)-Arg(Tos)-Gln-Met-Ala-Val-Arg(SEQID NO: 49) (Tos)-Arg(Tos)-Tyr(Cl₂-Bzl)-Leu-Asn-Ser(Bzl)-Ile-Leu-Asn-PAM.To the resulting protected peptide-PAM resin was added anhydroushydrogen fluoride in the presence of anisole to react them. After thereaction, anhydrous hydrogen fluoride was distilled off under reducedpressure, the residue was washed with ether, and 10% acetic acid wasadded thereto to extract the peptide. The extract was purified byreverse phase column chromatography, and lyophilized to obtain Peptide7.

Synthesis Example 19 Preparation of Peptide 8

Peptide 8 having an amino acid sequence shown in SEQ ID NO: 8 wassynthesized in the same manner as that of Peptide 5. Namely,coupling/deprotection was performed on a MBHA resin successively in anorder of Boc-Leu-OH, Boc-Ile-OH, Boc-Ser(Bzl)-OH, Boc-Asn(Xan)-OH,Boc-Leu-OH, Boc-Tyr(Cl₂-Z)-OH, Boc-Lys(Cl-Z)-OH, Boc-Lys(Cl-Z)-OH,Boc-Val-OH, Boc-Ala-OH, Boc-Met-OH, Boc-Gln(Xan)-OH, Boc-Lys(Cl-Z)-OH,Boc-Arg(Tos)-OH, Boc-Leu-OH, Boc-Arg(Tos)-OH, Boc-Thr(Bzl)-OH,Boc-Tyr(Cl₂-Z)-OH, Boc-Asn (Xan)-OH, Boc-Asp(OcHex)-OH, Boc-Thr(Bzl)-OH,Boc-Phe-OH, Boc-Val-OH, Boc-Ala-OH, Boc-Asp(OcHex)-OH, Boc-Ser(Bzl)-OH,and Boc-His(Bom)-OH, to obtain a protected peptide resin correspondingto Peptide 8; His(Bom)-Ser(Bzl)-Asp(OcHex)-Ala-Val-Phe-Thr(SEQ ID NO:45)(Bzl)-Asp(OcHex)-Asn-Tyr(Cl₂-Bzl)-Thr(Bzl)-Arg(Tos)-Leu-Arg(Tos)-Arg(Tos)-Gln-Met-Ala-Val-Arg(SEQID NO: 49) (Tos)-Arg(Tos)-Tyr(Cl₂-Bzl)-Leu-Asn-Ser(Bzl)-Ile-Leu-MBHA. Tothe resulting protected peptide-MBHA resin was added anhydrous hydrogenfluoride in the presence of anisole to react them. After the reaction,anhydrous hydrogen fluoride was distilled off under reduced pressure,the residue was washed with ether, and 10% acetic acid was added theretoto extract the peptide. The extract was purified by reverse phase columnchromatography, and lyophilized to obtain Peptide 8.

Synthesis Example 20 Preparation of Peptide 9

Peptide 9 having an amino acid sequence shown in SEQ ID NO: 9 wassynthesized in the same manner as that of Peptide 5. Namely,coupling/deprotection was performed on a MBHA resin successively in anorder of Boc-Ile-OH, Boc-Ser(Bzl)-OH, Boc-Asn(Xan)-OH, Boc-Leu-OH,Boc-Tyr(Cl₂-Z)-OH, Boc-Lys(Cl-Z)-OH, Boc-Lys(Cl-Z)-OH, Boc-Val-OH,Boc-Ala-OH, Boc-Met-OH, Boc-Gln(Xan)-OH, Boc-Lys(Cl-Z)-OH,Boc-Arg(Tos)-OH, Boc-Leu-OH, Boc-Arg(Tos)-OH, Boc-Thr(Bzl)-OH,Boc-Tyr(Cl₂-Z)-OH, Boc-Asn(Xan)-OH, Boc-Asp(OcHex)-OH, Boc-Thr(Bzl)-OH,Boc-Phe-OH, Boc-Val-OH, Boc-Ala-OH, Boc-Asp(OcHex)-OH, Boc-Ser(Bzl)-OHand Boc-His(Bom)-OH, to obtain a protected peptide resin correspondingto Peptide 9; His(Bom)-Ser(Bzl)-Asp(OcHex)-Ala-Val-Phe-Thr(SEQ ID NO:45)(Bzl)-Asp(OcHex)-Asn-Tyr(Cl₂-Bzl)-Thr(Bzl)-Arg(Tos)-Leu-Arg(Tos)-Arg(Tos)-Gln-Met-Ala-Val-Arg(SEQID NO: 49) (Tos)-Arg(Tos)-Tyr(Cl₂-Bzl)-Leu-Asn-Ser(Bzl)-Ile-MBHA. To theresulting protected peptide-MBHA resin was added anhydrous hydrogenfluoride in the presence of anisole to react them. After the reaction,anhydrous hydrogen fluoride was distilled off under reduced pressure,the residue was washed with ether, and 10% acetic acid was added theretoto extract the peptide. The extract was purified by reverse phase columnchromatography, and lyophilized to obtain Peptide 9.

Synthesis Example 21 Preparation of Peptide 10

Peptide 10 having an amino acid sequence shown in SEQ ID NO: 10 wassynthesized in the same manner as that of Peptide 5. Namely,coupling/deprotection was performed on a MBHA resin successively in anorder of Boc-Ser(Bzl)-OH, Boc-Asn(Xan)-OH, Boc-Leu-OH,Boc-Tyr(Cl₂-Z)-OH, Boc-Lys(Cl-Z)-OH, Boc-Lys(Cl-Z)-OH, Boc-Val-OH,Boc-Ala-OH, Boc-Met-OH, Boc-Gln(Xan)-OH, Boc-Lys(Cl-Z)-OH,Boc-Arg(Tos)-OH, Boc-Leu-OH, Boc-Arg(Tos)-OH, Boc-Thr(Bzl)-OH,Boc-Tyr(Cl₂-Z)-OH, Boc-Asn(Xan)-OH, Boc-Asp(OcHex)-OH, Boc-Thr(Bzl)-OH,Boc-Phe-OH, Boc-Val-OH, Boc-Ala-OH, Boc-Asp(OcHex)-OH, Boc-Ser(Bzl)-OH,and Boc-His(Bom)-OH, to obtain a protected peptide resin correspondingto Peptide 10; His(Bom)-Ser(Bzl)-Asp(OcHex)-Ala-Val-Phe-Thr(SEQ ID NO:45) (Bzl)-Asp (OcHex)-Asn-Tyr (Cl₂-Bzl)-Thr (Bzl)-Arg (Tos)-Leu-Arg(Tos)-Arg(Tos)-Gln-Met-Ala-Val-Arg(SEQ ID NO: 49) (Tos)-Arg(Tos)-Tyr(Cl₂-Bzl)-Leu-Asn-Ser (Bzl)-MBHA. To the resulting protectedpeptide-MBHA resin was added anhydrous hydrogen fluoride in the presenceof anisole to react them. After the reaction, anhydrous hydrogenfluoride was distilled off under reduced pressure, the residue waswashed with ether, and 10% acetic acid was added thereto to extract thepeptide. The extract was purified by reverse phase columnchromatography, and lyophilized to obtain Peptide 10.

Synthesis Example 22 Preparation of Peptide 11

Peptide 11 having an amino acid sequence shown in SEQ ID NO: 11 wassynthesized in the same manner as that of Peptide 5. Namely,coupling/deprotection was performed on a MBHA resin successively in anorder of Boc-Asn(Xan)-OH, Boc-Leu-OH, Boc-Tyr (Cl₂-Z)-OH,Boc-Lys(Cl-Z)-OH, Boc-Lys(Cl-Z)-OH, Boc-Val-OH, Boc-Ala-OH, Boc-Met-OH,Boc-Gln(Xan)-OH, Boc-Lys(Cl-Z)-OH, Boc-Arg(Tos)-OH, Boc-Leu-OH,Boc-Arg(Tos)-OH, Boc-Thr(Bzl)-OH, Boc-Tyr(Cl₂-Z)-OH, Boc-Asn(Xan)-OH,Boc-Asp(OcHex)-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH, Boc-Val-OH, Boc-Ala-OH,Boc-Asp(OcHex)-OH, Boc-Ser(Bzl)-OH, and Boc-His(Bom)-OH, to obtain aprotected peptide resin corresponding to Peptide 11;His(Bom)-Ser(Bzl)-Asp(OcHex)-Ala-Val-Phe-Thr(SEQ ID NO: 45)(Bzl)-Asp(OcHex)-Asn-Tyr(Cl₂-Bzl)-Thr(Bzl)-Arg(Tos)-Leu-Arg(Tos)-Arg(Tos)-Gln-Met-Ala-Val-Arg(SEQID NO: 49) (Tos)-Arg(Tos)-Tyr(Cl₂-Bzl)-Leu-Asn-MBHA. To the resultingprotected peptide-MBHA resin was added anhydrous hydrogen fluoride inthe presence of anisole to react them. After the reaction, anhydroushydrogen fluoride was distilled off under reduced pressure, the residuewas washed with ether, and 10% acetic acid was added thereto to extractthe peptide. The extract was purified by reverse phase columnchromatography, and lyophilized to obtain Peptide 11.

Synthesis Example 23 Preparation of Peptide 12

Peptide 12 having an amino acid sequence shown in SEQ ID NO: 12 wassynthesized in the same manner as that of Peptide 5. Namely,coupling/deprotection was performed on a MBHA resin successively in anorder of Boc-Leu-OH, Boc-Tyr(Cl₂-Z)-OH, Boc-Lys(Cl-Z)-OH,Boc-Lys(Cl-Z)-OH, Boc-Val-OH, Boc-Ala-OH, Boc-Met-OH, Boc-Gln(Xan)-OH,Boc-Lys(Cl-Z)-OH, Boc-Arg(Tos)-OH, Boc-Leu-OH, Boc-Arg(Tos)-OH,Boc-Thr(Bzl)-OH, Boc-Tyr (Cl₂-Z)-OH, Boc-Asn(Xan)-OH, Boc-Asp(OcHex)-OH,Boc-Thr(Bzl)-OH, Boc-Phe-OH, Boc-Val-OH, Boc-Ala-OH, Boc-Asp(OcHex)-OH,Boc-Ser(Bzl)-OH, and Boc-His(Bom)-OH, to obtain a protected peptideresin corresponding to Peptide 12;His(Bom)-Ser(Bzl)-Asp(OcHex)-Ala-Val-Phe-Thr(SEQ ID NO: 45)(Bzl)-Asp(OcHex)-Asn-Tyr(Cl₂-Bzl)-Thr(Bzl)-Arg(Tos)-Leu-Arg(Tos)-Arg(Tos)-Gln-Met-Ala-Val-Arg(SEQID NO: 49) (Tos)-Arg(Tos)-Tyr(Cl₂-Bzl)-Leu-MBHA. To the resultingprotected peptide-MBHA resin was added anhydrous hydrogen fluoride inthe presence of anisole to react them. After the reaction, anhydroushydrogen fluoride was distilled off under reduced pressure, the residuewas washed with ether, and 10% acetic acid was added thereto to extractthe peptide. The extract was purified by reverse phase columnchromatography, and lyophilized to obtain Peptide 12.

Synthesis Example 24 Preparation of Peptide 13

Peptide 13 having an amino acid sequence shown in SEQ ID NO: 13 wassynthesized in the same manner as that of Peptide 5. Namely,coupling/deprotection was performed on a MBHA resin successively in anorder of Boc-Ser(Bzl)-OH, Boc-Asn(Xan)-OH, Boc-Leu-OH,Boc-Tyr(Cl₂-Z)-OH, Boc-Lys(Cl-Z)-OH, Boc-Lys(Cl-Z)-OH, Boc-Val-OH,Boc-Ala-OH, Boc-Leu-OH, Boc-Gln(Xan)-OH, Boc-Lys(Cl-Z)-OH,Boc-Arg(Tos)-OH, Boc-Leu-OH, Boc-Arg(Tos)-OH, Boc-Thr(Bzl)-OH,Boc-Tyr(Cl₂-Z)-OH, Boc-Asn(Xan)-OH, Boc-Asp(OcHex)-OH, Boc-Thr(Bzl)-OH,Boc-Phe-OH, Boc-Val-OH, Boc-Ala-OH, Boc-Asp(OcHex)-OH, Boc-Ser(Bzl)-OH,and Boc-His(Bom)-OH, to obtain a protected peptide resin correspondingto Peptide 13; His(Bom)-Ser(Bzl)-Asp(OcHex)-Ala-Val-Phe-Thr(SEQ ID NO:45) (Bzl)-Asp (OcHex)-Asn-Tyr (Cl₂-Bzl)-Thr (Bzl)-Arg (Tos)-Leu-Arg(Tos)-Arg(Tos)-Gln-Leu-Ala-Val-Arg(SEQ ID NO: 46)(Tos)-Arg(Tos)-Tyr(Cl₂-Bzl)-Leu-Asn-Ser(Bzl)-MBHA. To the resultingprotected peptide-MBHA resin was added anhydrous hydrogen fluoride inthe presence of anisole to react them. After the reaction, anhydroushydrogen fluoride was distilled off under reduced pressure, the residuewas washed with ether, and 10% acetic acid was added thereto to extractthe peptide. The extract was purified by reverse phase columnchromatography, and lyophilized to obtain Peptide 13.

Synthesis Example 25 Preparation of Peptide 14

Peptide 14 having an amino acid sequence shown in SEQ ID NO: 14 wassynthesized in the same manner as that of Peptide 5. Namely,coupling/deprotection was performed on a MBHA resin successively in anorder of Boc-Asn(Xan)-OH, Boc-Leu-OH, Boc-Tyr (Cl₂-Z)-OH,Boc-Lys(Cl-Z)-OH, Boc-Lys(Cl-Z)-OH, Boc-Val-OH, Boc-Ala-OH, Boc-Leu-OH,Boc-Gln(Xan)-OH, Boc-Lys(Cl-Z)-OH, Boc-Arg(Tos)-OH, Boc-Leu-OH, Boc-Arg(Tos)-OH, Boc-Thr (Bzl)-OH, Boc-Tyr (Cl₂-Z)-OH, Boc-Asn(Xan)-OH,Boc-Asp(OcHex)-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH, Boc-Val-OH, Boc-Ala-OH,Boc-Asp(OcHex)-OH, Boc-Ser(Bzl)-OH, and Boc-His(Bom)-OH, to obtain aprotected peptide resin corresponding to Peptide 14;His(Bom)-Ser(Bzl)-Asp(OcHex)-Ala-Val-Phe-Thr(SEQ ID NO: 45)(Bzl)-Asp(OcHex)-Asn-Tyr(Cl₂-Bzl)-Thr(Bzl)-Arg(Tos)-Leu-Arg(Tos)-Arg(Tos)-Gln-Leu-Ala-Val-Arg(SEQID NO: 46) (Tos)-Arg(Tos)-Tyr(Cl₂-Bzl)-Leu-Asn-MBHA. To the resultingprotected peptide-MBHA resin was added anhydrous hydrogen fluoride inthe presence of anisole to react them. After the reaction, anhydroushydrogen fluoride was distilled off under reduced pressure, the residuewas washed with ether, and 10% acetic acid was added thereto to extractthe peptide. The extract was purified by reverse phase columnchromatography, and lyophilized to obtain Peptide 14.

Preparation Example 1

Eye Drops 1 to be Dissolved Upon Use

Peptide 5    2 g Sodium chloride  0.9 g Boric acid  0.1 g Borax q.s. (pH7.8) Benzalkonium chloride 0.005 g Sodium edetate  0.02 g Purified waterad. 100 mL

In 100 mL of purified water, 5 g of Peptide 5 is dissolved and filteredthrough a membrane filter (0.45 μm). Each 2 mL portion of this solutionis dispensed into a 5 mL-eye drop container, and lyophilized. In about80 mL of purified water, 0.9 g of sodium chloride, 0.1 g of boric acid,0.005 g of benzalkonium chloride and 0.02 g of sodium edetate aredissolved, and the pH is adjusted to 7.8 with borax, and then, a totalvolume is adjusted to 100 mL with purified water. This solution isfiltered through a membrane filter (0.45 μm), and dispensed into a 5 mLampoule and sealed to obtain a dissolving solution. Upon use, thedissolving solution is poured into the above 5 mL-eye drop container toobtain eye drops.

Preparation 2 Eye Drops 2 to be Dissolved Upon Use

Peptide 15  0.1 g Mannitol    5 g Boric acid  0.1 g Borax q.s. (pH 7.8)Benzalkonium chloride 0.005 g Sodium edetate  0.02 g Purified water ad.100 mL

In 100 mL of purified water, 0.25 g of Peptide 15 and 12.5 g of mannitolare dissolved, and filtered through a membrane filter (0.45 μm). Each 2mL portion of this solution is dispensed into a 5 mL-eye drop container,and lyophilized. In about 80 mL of purified water, 0.1 g of boric acid,0.005 g of benzalkonium chloride and 0.02 g of sodium edetate aredissolved, and the pH is adjusted to 7.8 with borax, and a total volumeis adjusted to 100 mL with purified water. This solution is filteredthrough a membrane filter (0.45 μm), and dispensed into a 5 mL ampouleand sealed to obtain a dissolving solution. Upon use, the dissolvingsolution is poured into the above 5 mL-eye drop container to obtain eyedrops.

Preparation Example 3 One-Pack Type Eye Drops 1

Peptide 5  0.1 g Boric acid  0.7 g Borax q.s. (pH 7.7) Sodium chloride 0.5 g Sodium edetate  0.05 g Benzalkonium chloride 0.005 g Purifiedwater ad. 100 mL

Sodium chloride, boric acid, sodium edetate and benzalkonium chlorideare added to about 80 mL of purified water and dissolved therein, andthe pH is adjusted to 7.7 by adding borax. Peptide 5 is added to thissolution and dissolved therein, and then, a total volume is made up to100 mL with purified water to obtain eye drops.

Preparation Example 4 One-Pack Type Eye Drops 2

Peptide 15  0.5 g Sodium chloride  0.9 g Sodium dihydrogen phosphatedihydrate  0.1 g Benzalkonium chloride 0.005 g 0.1 N sodium hydroxideq.s. (pH 7.2) Purified water ad. 100 mL

Sodium chloride, sodium dihydrogen phosphate.dihydrate and benzalkoniumchloride are added to about 80 mL of purified water and dissolvedtherein, and the pH is adjusted to 7.2 by adding 0.1 N sodium hydroxide.Peptide 15 is added to this solution and dissolved therein, and then, atotal volume is made up to 100 mL with purified to obtain eye drops.

Preparation Example 5 Injectable Preparation 1

In water for injection, 1.0 mg of Peptide 5, 200 mg of sodium chlorideand 300 mg of gelatin are dissolved. An appropriate amount of a pHadjusting agent is added thereto so as to adjust pH to 7.4, a totalvolume is made up to 20 mL with water for injection, and the solution issterilized by filtration, each 2 mL portion of which is dispensed into aglass ampoule.

Preparation Example 6 Injectable Preparation 2

In water for injection, 1.0 mg of Peptide 15, 200 mg of sodium chlorideand 250 mg of albumin are dissolved. An appropriate amount of a pHadjusting agent is added thereto so as to adjust pH to 7.4, a totalvolume is made up to 20 mL with water for injection, and the solution issterilized by filtration, each 2 mL portion of which is dispensed into aglass ampoule.

Test Example 1 Trypsin Digestion Test

The effect of highly basified Peptide 5 on digestion with trypsin wasstudied in the presence of chondroitin sulfate.

1) Test Method

To 100 μL of a solution of VIP and Peptide 5 (1 mg/mL) was added 400 μLof a solution of chondroitin sulfate (pH 7.5, 100 μg/mL) in a 0.2 MTris-HCl buffer (pH 7.5). To 100 μL of this solution was added 0.5 mU oftrypsin and the mixture was incubated at 37° C. for 180 minutes. Then,an amount of an undegraded peptide was measured by HPLC.

2) Results

In digestion by trypsin in the presence of chondriotin sulfate, theremaining rate of VIP was 75%, while that of Peptide 5 was as high as89%. This result suggests that interaction of this peptide and acidicpolysaccharides exhibits resistance to enzymatic degradation.

Test Example 2 Digestion Test with Bronchoalveolar Lavage Fluid

The effect of highly basified Peptide 5 on digestion withbronchoalveolar lavage fluid was studied.

1) Test Method

VIP and Peptide 5 were dissolved (1.1 mg/mL) in a 0.2% Tris-HCl buffer(pH 7.4), and bronchoalveolar lavage fluid (BALF) was added thereto.BALF was obtained by pouring 5 mL of a physiological saline into atrachea of a 8 to 9 week old normal SD male rat (about 350 g) underanesthesia, and recovering the saline, and repeating this proceduretotally three times. To 0.5 mL of BALF was added 0.05 mL of a samplesolution and the mixture was reacted at 37° C. for 90 minutes, andthereafter an amount of undegraded peptide was measured by HPLC.

2) Results

In a digestion test with BALF, the remaining rate of VIP was 45.5%,while the remaining rate of Peptide 5 was 80.0%, demonstrating about1.8-fold higher remaining rate as compared with VIP. This resultsuggests that this peptide exhibits the excellent stability in a livingbody.

Test Example 3 Activity of Promoting Secretion of Protein from IsolatedLacrimal Grand

An isolated lacimal gland of a rabbit was treated with VIP, or Peptide 5or 15, and the pharmacological effect of these peptides on increase inan amount of secreted protein was studied.

1) Preparation of Test Solution

a) Purified water was added to sodium chloride (137.92 g), potassiumchloride (7.01 g), calcium chloride-dihydrate (3.53 g) and magnesiumchloride.hexahydrate (1.2 g) and the mixture was made up to a totalvolume of 1000 mL (Solution 1). Separately, purified water was added topotassium dihydrogen phosphate (8.17 g) and the mixture was made up to atotal volume of 500 mL (Solution 2). Solution 1 (50 mL) and Solution 2(10 mL) were added to 900 mL of purified water, 2.07 g of glucose and2.1 g of sodium dicarbonate were dissolved therein, a total amount wasadjusted to 1000 mL (pH about 7.4) to obtain an incubation medium. Theincubation medium was bubbled with mixed gas containing 95% oxygen and5% carbon dioxide.

b) VIP, Peptide 5 or Peptide 15 was dissolved in the incubation mediumat a concentration of 10⁻⁴ M to obtain a test solution.

2) Preparation of Lacrimal Gland Piece Specimen

A male Japanese white rabbit was systemically anesthetized, a perfusionsolution (containing 116 mM sodium chloride, 5.4 mM potassium chloride)was perfused through an abdominal aorta, a main lacrimal gland tissuewas isolated, and a fat connective tissue was removed, which was dividedequally (one piece was about 40 mg). This lacrimal gland tissue piecewas transferred to a 24-well plate wherein each well thereof was filledwith 0.5 mL of the incubation medium, followed by incubation at 37° C.The incubation medium was exchanged every 20 minutes three times,allowed to stand for a total of 60 minutes, to obtain a lacrimal glandpiece specimen in the steady state.

3) Test Method

a) The piece specimen in the steady state was used, the incubationmedium was exchanged with 0.5 mL of a fresh incubation medium, followedby incubation at 37° C. for 20 minutes. This incubation medium wascollected, a protein staining reagent (DC Protein assay reagent, BioRad) was added thereto, and an amount of protein was measured andexpressed as an amount of secreted protein per mg of wet weight. Anamount of protein thereupon was regarded as protein secretion rate 100%,and used as a secretion rate before treatment.

b) The incubation medium of the piece specimen after incubation in a)was exchanged with 0.5 mL of a test solution, and the solution wasexchanged and collected at 20 minutes intervals totally five times. Aprotein staining reagent was added to the collected solution, and anamount of protein was measured. For a piece specimen in a control group,the incubation medium was exchanged similarly, and an amount of proteinwas measured.

4) Results

The test results are shown in FIG. 1. A vertical axis indicates a rate(%) of protein secretion from isolated lacrimal gland, and a horizontalaxis indicates a time (min.) of treatment with each test solution. Whentreated with VIP, Peptide 5 and Peptide 15, a rate of protein secretionwas significantly increased for a treating time of 0 to 40 minutes ascompared with a control group (n=5, mean±standard error, *; p<0.05, **;p<0.01, Dunnett's test). Namely, it has been revealed that all ofPeptides 5 and 15 and VIP have the excellent protein secretion promotingactivity.

Test Example 4 Lacrimal Secretion Promotion Activity of Eye Drops (1)

Peptide 5 or 15 was applied to eyes of a rabbit, and an amount ofsecreted tears was measured, whereby, the pharmacological effect ofthese peptides on increase in an amount of lacrimal secretion wasstudied.

1) Preparation of Solution

Purified water was added to sodium chloride (0.9 g) and sodiumdihydrogen phosphate.dihydrate (0.1 g) to make up to a total volume of100 mL. Further, an appropriate amount of aqueous sodium hydroxidesolution was added so as to adjust pH to 7.0 to obtain a vehicle.Peptide 5 was dissolved in the vehicle at a concentration of 0.1 w/v %,and Peptide 15 was dissolved in the vehicle at a concentration of 2.0w/v %, to prepare test solutions.

2) Test Method

Measurement of an amount of tears was performed by a Schirmer test. Toeyes of a Japanese white rabbit, 50 μL of a test solution was appliedonce, and amounts of lacrimal secretion before and 10 minutes after theapplication to eyes were measured. Five minutes before measurement of anamount of tears, 10 μL of 0.4 w/v % oxybuprocaine hydrochloride(Anelocal™ eye drops, Senju Pharmaceutical Co., Ltd.) was dropped totopically anesthetize. After tears in palpebra interior conjunctival sacwere wiped out using a filter paper, and an amount of lacrimal secretionfor 1 minute was measured using a Schirmer test paper (Showa YakuhinKako Co., Ltd.).

3) Results

The test results are shown in Table 2. By single application to eyes ofa rabbit, an amount of lacrimal secretion was significantly increased inapplication of the Peptide 5 solution (*; p<0.01, paired t test). Inaddition, in application of the Peptide 15 solution, a statisticallysignificant difference was not recognized, but a tendency of increasewas shown (p=0.13, paired t test).

Namely, it has been revealed that Peptides 5 and 15 have excellentlacrimal secretion promotion activity.

TABLE 2 Amount of lacrimal secretion (Schirmer's value: mm/min, n = 6,mean ± standard error) Before After administration administration 0.1%Peptide 5 2.00 ± 0.62 8.92 ± 0.45* 2.0% Peptide 15 3.25 ± 1.20 7.75 ±2.16 

Test Example 5 Lacrimal Secretion Promotion Activity of Eye Drops (2)

In the same manner as that of Test Example 4, Peptide 5 was applied, andan amount of lacrimal secretion was measured. In this test, an amount oflacrimal secretion was measured with time, and the effect was comparedwith that of VIP.

1) Preparation of Solution

Preparation of a solution was performed in the same manner as that ofTest Example 4, and VIP and Peptide 5 were dissolved in the vehicle at aconcentration of 0.1 w/v %, to prepare a test solution.

2) Test Method

50 μL of the vehicle or a test solution was applied to eyes of aJapanese white rabbit once, and amounts of lacrimal secretion weremeasured before and 10 minutes, 20 minutes, 30 minutes, 60 minutes and100 minutes after the application. Measurement of an amount of lacrimalsecretion was performed in the same manner as that of Test Example 4.

3) Results

A change in an amount of lacrimal secretion after administration isshown in FIG. 2. A vertical axis indicates a difference (increment) inan amount of lacrimal liquid secretion (Schirmer's value: mm/min.) fromthat before administration. A horizontal axis indicates passage of time(min.). By application of VIP and Peptide 5 to eyes, the amount wassignificantly increased at 10 minutes after the administration ascompared with a control group of base administration (n=8, mean±standarderror, *; p<0.05, **; p<0.01, parametric Dunnett-type multiplecomparison test). An accumulated value of an increment of an amount oflacrimal secretion for 120 minutes after the administration (AUCvalue=ΔSchirmer's value×min.) is shown in Table 3. The AUC value is inan order of Peptide 5 administered group>VIP administered group>controlgroup, and a Peptide 5 applied group shows a significantly higher valueas compared with a control group (*; p<0.05). On the other hand, in aVIP applied group, a significant difference is not recognized ascompared with a control group (p=0.38). Namely, it has been revealedthat Peptide 5 has the excellent lacrimal secretion promoting activityas compared with VIP.

TABLE 3 Accumulated value of increment of amount of lacrimal secretionafter application AUC_(0-120 min) (ΔSchirmer's Administered group value× min.) Vehicle administered group 65.6 ± 72.9 (control group) 0.1% VIPadministered group 215.0 ± 98.2  0.1% Peptide 5 administered group 359.2± 85.0* n = 8, average ± standard error *p < 0.05, parametricDunnett-type multiple comparison test

INDUSTRIAL APPLICABILITY

The above results demonstrate that the VIP derivative of the presentinvention has the excellent lacrimal secretion promoting activity, andis useful as a remedy for dry eye and diseases associated with dry eye.

1. A method for treating dry eye or dry eye associated with disease,comprising topically administering to an eye of a warm-blooded animal inneed thereof an eye drop comprising 0.01-0.5% w/v % of a peptideselected from the group consisting of:His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Arg-Gln-Leu-Ala-Val-Arg-Arg-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-Gly(SEQ ID NO:1),His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Arg-Gln-Leu-Ala-Val-Arg-Arg-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-Gly-Lys(SEQ ID NO:2),His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Arg-Gln-Leu-Ala-Val-Arg-Arg-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-Gly-Arg(SEQ ID NO:3),His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Arg-Gln-Leu-Ala-Val-Arg-Arg-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-Gly-Lys-Arg(SEQ ID NO:4),His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Arg-Gln-Leu-Ala-Val-Arg-Arg-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-Gly-Arg-Arg(SEQ ID NO:5), andHis-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Arg-Gln-Leu-Ala-Val-Arg-Arg-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-Gly-Lys-Arg(SEQ ID NO:6) wherein the C-terminus is optionally amidated with NH2, ora pharmaceutically acceptable salt thereof.
 2. The method of claim 1,wherein the peptide is SEQ ID NO:5 and the C-terminus is amidated.